Name: murine model of cd40-activation of b cells
Uploaded: 2010-03-05T17:30:09
Description: Watch this Scientific Journal Video about Murine Model of CD40-activation of B cells at JoVE.com

我们诚恳地感谢克莱门斯Wendtner博士提供tmuCD40配体HeLa细胞株。

协议分为两个部分：第一部分为一代小鼠CD40激活的B细胞（脾mCD40B）一个演示小鼠CD40配体（CD40L）表达HeLa细胞（tmuCD40L HeLa细胞），这将被用作板准备束缚饲养层细胞。 B部分介绍了实际的小鼠CD40 - B的文化。 A.制备饲养细胞（HeLa细胞tmuCD40L） tmuCD40L HeLa细胞是一个附着的人类上皮细胞系，这不应该成为完全融合。因此，这些细胞被劈裂每周两次。超过6周以...

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	<li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+human+B+cells:+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy.">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> J Clin Invest. 100 (11), 2757-2765 (1997).</li><li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+human+B+cells+by+CD40+ligation.">Conditional immortalization of human B cells by CD40 ligation.</a> PLoS ONE. 3, e1464-e1464 (2008).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+antigen-specific+cytotoxic+T+cells+using+retrovirally+transduced+CD40-activated+B+cells.">Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells.</a> J Immunol. 169 (4), 2164-2171 (2002).</li><li>Liebig, T. M., Fiedler, A., Zoghi, S., Shimabukuro-Vornhagen, A., Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+CD40-activated+B+cells.">Generation of human CD40-activated B cells.</a> J Vis Exp. 32, (2009).</li><li>von Bergwelt-Baildon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis:+potential+as+cellular+adjuvants.">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> Blood. 107 (7), 2786-2789 (2006).</li><li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells.">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> Cancer Res. 63 (11), 2836-2843 (2003).</li><li>Ahmadi, T., Flies, A., Efebera, Y., Sherr, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+Ligand-activated,+antigen-specific+B+cells+are+comparable+to+mature+dendritic+cells+in+presenting+protein+antigens+and+major+histocompatibility+complex+class+I-+and+class+II-binding+peptides.">CD40 Ligand-activated, antigen-specific B cells are comparable to mature dendritic cells in presenting protein antigens and major histocompatibility complex class I- and class II-binding peptides.</a> Immunology. 124 (1), 129-140 (2008).</li><li>Bergwelt-Baildon, M. S. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells:+potential+for+clinical+application.">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> Blood. 99 (9), 3319-3325 (2002).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients:+analysis+of+immunogenicity+and+homing+potential.">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> Clin Exp Immunol. 155 (2), 249-256 (2009).</li><li>Schultze, J. L., Grabbe, S., von Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+and+CD40-activated+B+cells:+current+and+future+avenues+to+cellular+cancer+immunotherapy.">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> Trends Immunol. 25 (12), 659-664 (2004).</li><li>Mayr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+translocations+are+associated+with+poor+prognosis+in+chronic+lymphocytic+leukemia.">Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia.</a> Blood. 107 (2), 742-751 (2006).</li><li>Mason, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma.">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> Gene Ther. 15 (13), 955-965 (2008).</li></ol>

本协议的修改是可能的，尤其是关于CD40的刺激和使用的小鼠品系类型的起源，也高产高效代小鼠CD40激活的B细胞。艾哈迈迪等人。表明小鼠CD40的配体转染小鼠成纤维细胞类似的结果。可以肯定地排除在这种异种抗原的背景下演示，但在体内的安全性和毒性的深入研究了在此设置使用人类转染的HeLa细胞的炎症或自身免疫反应的迹象。然而，最好的替代代表与激活和增殖活动在同一时间，这?...

A.媒体的制备： <table border="1"><tbody><tr><td> 直属细胞野生型中 </td><td> 馈线小区选择培养基 </td><td></td></tr><tr><td> 试剂 </td><td> 集中 </td><td> 试剂 </td><td> 集中 </td></tr><tr><td> RPMI 1640培养</td><td></td><td> RPMI 1640培养</td><td></td></tr><tr><td> L -谷氨酰胺</td><td> 300微克/毫升</td><td> L -谷氨酰胺</td><td> 300微克/毫升</td></tr><tr><td>胎牛血清</td><td> 10％ </td><td>胎牛血清</td><td> 10％ </td></tr><tr><td> HEPES </td><td> 10毫米</td><td> HEPES </td><td> 10毫米</td></tr><tr><td>庆大霉素</td><td> 15微克/毫升</td><td>庆大霉素</td><td> 15微克/毫升</td></tr><tr><td></td><td></td><td>潮霉素B </td><td> 0.2毫克/毫升。 </td></tr></table><table border="1"><tbody><tr><td> mCD40 - B的清洗介质 </td><td> mCD40 - B的培养基 </td></tr><tr><td> 试剂 </td><td> 集中 </td><td> 试剂 </td><td> 集中 </td></tr><tr></tr><tr><td> D - MEM </td><td> </td><td> D - MEM </td><td> </td></tr><tr><td> L -谷氨酰胺</td><td> 580微克/毫升</td><td> L -谷氨酰胺</td><td> 580微克/毫升</td></tr><tr><td>血糖</td><td> 4.5毫克/毫升</td><td>血糖</td><td> 4.5毫克/毫升</td></tr><tr><td> HEPES </td><td> 10毫米</td><td> HEPES </td><td> 10毫米</td></tr><tr><td>庆大霉素</td><td> 15微克/毫升</td><td>庆大霉素</td><td> 15微克/毫升</td></tr><tr><td> </td><td> </td><td>纪念</td><td> 0.1MM（1X） </td></tr><tr><td> </td><td> </td><td>胎牛血清</td><td> 10％ </td></tr></tbody></table> B.其它试剂： <table border="1"><tr><td> 试剂 </td><td> 公司 </td><td> 订单号 </td></tr><tr><td> RPMI 1640培养</td><td> Invitrogen公司Gibco公司</td><td>楼盘21875 </td></tr><tr><td> D - MEM </td><td> Invitrogen公司Gibco公司</td><td>楼盘41965 </td></tr><tr><td> Dulbeccos PBS（10X） </td><td>临时机场管理局</td><td>猫没有H15 - 011 </td></tr><tr><td> Gencin </td><td>三角洲选择</td><td>货号7395800 </td></tr><tr><td>胎牛血清</td><td>龙沙</td><td>猫没有DE14 - 802C </td></tr><tr><td> HEPES缓冲液</td><td>临时机场管理局</td><td>猫没有S11 - 001 </td></tr><tr><td> MEM NEAA </td><td> Invitrogen公司Gibco公司</td><td>楼盘11140 </td></tr><tr><td>潮霉素B </td><td>临时机场管理局</td><td>猫没有P02 - 015 </td></tr><tr><td>重组小鼠白细胞介素4 </td><td> Immunotools </td><td> CAT无12340045 </td></tr><tr><td>环孢素A </td><td>诺华公司</td><td>猫没有NDC 0078-0109-01 </td></tr><tr><td>胰蛋白酶/ EDTA（10X） </td><td> Invitrogen公司Gibco公司</td><td>楼盘15400-054 </td></tr></table> C.杂项用品： <table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 订单号 </td></tr><tr><td>无菌移液器</td><td> Sarstedt </td><td></td></tr><tr><td> 6孔板</td><td> NUNC </td><td> CAT没有140675 </td></tr><tr><td> 50毫升锥形管</td><td> BD猎鹰™ </td><td> CAT没有352070 </td></tr><tr><td>组织培养瓶</td><td> Sarstedt </td><td> CAT没有83.1813.002 </td></tr></tbody></table>

murine model of cd40-activation of b cells

有研究表明，B细胞CD40的激活提高其抗原提呈能力。当白细胞介素4和CD40配体（CD40L）的刺激下，没有非常大量高纯度的CD40 - B细胞（14天之内的困难，从少量外周血的可扩展人类B细胞&gt; 10<sup&gt; 9</sup&gt;细胞每名患者），从健康的捐赠者以及癌症患者<sup&gt; 1-4</sup&gt;。 CD40 - B细胞表达重要的淋巴结归巢的分子，可以吸引T细胞<em&gt;在体外</em<sup&gt; 5</sup&gt;。此外，他们采取有效，过程和目前的T细胞抗原<sup&gt; 6,7</sup&gt;。 CD40 - B细胞被证明不仅总理天真，但也扩大记忆性T细胞<sup&gt; 8,9</sup&gt;。因此，CD40 -激活的B细胞（CD40 - B细胞）进行了研究，作为一种替代源免疫刺激抗原提呈细胞（APC），基于细胞immunotherapy1，5,10。为了进一步研究是否CD40 - B细胞诱导有效的T细胞在体内的反应和我们建立了小鼠CD40激活的B细胞产生的细胞培养系统研究的基础机制。使用脾细胞或纯化C57BL / 6小鼠的B细胞CD40的激活，确定了最佳工艺条件如下：开始的C57BL / 6小鼠淋巴细胞（单体型H - 2B）的脾细胞密度梯度离心法和HeLa细胞共同培养纯化重组鼠CD40配体（tmuCD40L的HeLa细胞表达）<sup&gt; 11</sup&gt;。细胞是再培养每隔3-4天，和关键部件，如CD40L，白细胞介素- 4 -巯基乙醇和环孢素A的补充。在这个协议中，我们演示了如何获得类似的APC -表型的人CD40 B细胞（图1A，B）完全激活的小鼠的CD40 - B细胞（mCD40B）。 CD40的刺激会导致快速的生长和细胞培养在14天的高纯度（&gt; 90％），CD19 + B细胞的扩张（图1C，D）​​。为了避免污染与非转染的细胞，小鼠CD40的配体的转染表达经常被控制（图2）。小鼠CD40激活的B细胞可用于研究B细胞的活化和分化，以及调查他们的潜能作为APC，在体内和体外的功能。此外，他们代表了建立抗肿瘤治疗或预防接种有前途的工具，将有助于回答问题，这种方法的安全性和免疫原性<sup&gt; 12</sup&gt;。

Watch this Scientific Journal Video about Murine Model of CD40-activation of B cells at JoVE.com

Murine Model of CD40-activation of B cells

在这个视频中，我们展示的CD40从C57BL / 6小鼠的脾细胞，它可以用来作为模型抗原提呈细胞（APC）的研究免疫诱导小鼠的B细胞的活化和扩大的过程。

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Research

JoVE Journal

Immunology and Infection

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

纯净小鼠的B细胞的诱导和评估类开关重组

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

科研

免疫与感染

Recombinant Retroviral Production and Infection of B Cells

重组逆转录病毒的生产和B细胞的感染

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Murine Model of Allergen Induced Asthma

过敏原诱发的哮喘小鼠模型

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Plaque Assay for Murine Norovirus

对小鼠诺沃克类病毒的空斑法

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Detection of True IgE-expressing Mouse B Lineage Cells

检测真正的IgE的表达小鼠B​​系细胞

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

A Murine Model of Group B Streptococcus Vaginal Colonization

A Murine Model of Group B Streptococcus Vaginal Colonization

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

小鼠边缘区 b 细胞剪切流诱导迁移的体外分析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

人正常记忆 b 细胞与长寿浆细胞体外分化模型

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...